Flavoproteins catalyze many important biological redox reactions. Understanding the mechanism of reaction is of primary importance. Because the redox potential (E') is a measure of the equilibrium energy level of the enzyme, it is important and will serve as a reference for further studies. A previous study indicates the protein matrix regulates both the redox properties and the catalytic activities of the flavin. In support of this idea the redox data, kinetic data, and structural data have been obtained for flavodoxin and glucose oxidase. Taken together this data has provided important insights into flavoprotein mechanisms. These studies stress the need for a very detailed study of redox potentials of the flavoproteins. Despite the importance of the redox properties E' and n (number of electrons transferred), no systematic study of flavoprotein redox properties has previously been made. We developed a new spectroelectrochemical method for studying the redox properties of flavoproteins. By using this method we were able to obtain the first measurements of E' and n for the flavoprotein oxidases. The redox data correlates very well with newly available structural data on these enzymes. However, it is difficult to correlate the redox data with the known catalytic mechanism for these enzymes. We now suggest a way in which the two can be related. We propose that binding of substrate to the enzyme further "tunes" the energy levels of the enzymes. If this hypothesis is correct, the E' and n values of substrate and inhibitor bound enzymes should be different from the E' and n values of the free enzymes which we have already measured. We propose to test our hypothesis by measuring the redox properties (E' and n) of both oxidases and members of the other flavoprotein classes bound to substrates and inhibitors. We will continue our systematic measurement of the free flavoprotein redox properties. Enzyme regulation by substrate binding has been determined for cytochrome P450 cam. That regulation was identified by redox potential measurements of free and substrate bound enzyme. Our own work has shown that the binding of competitive inhibitors to D-amino acid oxidase significantly alters both E' and n. Thus, we are addressing basic questions about enzyme regulation. The electrochemical method is the best method of providing answers to these questions.